The proposed work aims to develop a pre-commercial prototype of an exosome isolation device to be used in clinical diagnostic laboratories. We will advance the device to facilitate exosome isolation for downstream liquid biopsy tests requiring analysis of protein, DNA, or microRNA, which rely upon acquisition of intact and pure exosomes. The device strategy will be designed to isolate total exosomes and then to separate discrete populations of tumor-derived exosomes (TEX) and non-malignant exosomes (non-TEX). The device strategy will be advanced for melanoma exosome isolation in the proposed experiments but is expected to be applicable for other cancers. To achieve this study's objectives, we will develop and optimize a filtration system incorporating the GNA Hemopurifier for isolation of total exosomes from biofluids (Specific Aim 1). The capabilities of the GNA Hemopurifier for isolating melanoma TEX and non-TEX from diverse biofluids will be characterized by evaluating exosome depletion performance (Specific Aim 2). We will then construct a novel device, the CSPG4 cartridge, for the isolation of TEX from total exosomes, followed by testing the final workflow from the starting biofluid sample to the final TEX isolation (Specific Aim 3). Successful completion of this project will produce an exosome isolation system that will be applicable for high throughput exosome isolation and for TEX and non-TEX separation from diverse biofluids of varying complexity in a clinical care setting in oncology.